Xenbase Image ID: 81902

Fig. 5. Over-expression of PACSIN2 prevents cell spreading and perturbs convergence and extension. (A, B) Animal cap ectoderm of non injected embryos (NI) or embryos over-expressing the wild type (Wt) or the mutant form of PACSIN2 (∂CC) were dissociated at stage 8 and the cells seeded on GST fusion protein containing the CCBD domain of FN, in the presence (+ Activin) or absence of Activin (− Activin). Photographs were taken after sibling embryos initiated gastrulation movements. (A) Control cells (NI) or cells expressing ∂CC (∂CC) spread on the substrate in the presence of Activin-A. The cells over-expressing Wt-PACSIN2 (Wt) do not spread in those conditions. In the absence of Activin-A, the cells do not spread. The percentage of spread cells after Activin-A treatment was plotted on the histogram. The graph represent the mean of four independent experiments and error bars represent the standard deviation from the mean. The number of cells analyzed are as follow: NI = 559; Wt = 627; ∂CC = 717. Arrows: filopodia; Arrowhead: lamelipodia. (B) Cells over-expressing Wt-PACSIN2 were treated as previously described. After 2 h, cells were treated with 0.5 mM of MnCl2. Pictures were taken just before the addition of MnCl2 and 30 min after. The percentage of spread cells was plotted on the histogram. The graph represent the mean of 2 independent experiments and errors bars represent the standard deviation from the mean. The number of cells analyzed were as followed: NI = 269; NI + Mn2+ = 332; Wt = 292; Wt + Mn2+ = 206. The activation of integrins by Mn2+ is sufficient to rescue the spreading of the cells over-expressing Wt PACSIN2 (P < 0.05). (C) Animal cap from non injected embryos (NI) or from embryos expressing Wt or ∂CC PACSIN2 were dissected at stage 8, induced with 5 ng/ml of Activin-A and grown until the sibling embryos reached stage 20 (Ctl). While all animal caps from non-injected embryos or caps expressing ∂CC extend in response to Activin treatment, elongation is dramatically reduced in the animal caps over-expressing Wt-PACSIN2. The histogram represents the average length of explants in arbitrary units in a representative experiment. Three independent experiments were performed with similar results. The numbers of explants analyzed are as follow: NI = 37; Wt = 22; ∂CC = 14. ⁎: P < 0.05. Image published in: Cousin H et al. (2008) Copyright © 2008. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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